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PURPOSEPatients with relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) have few options and poor prognosis. The aim was to assess donor-derived anti-CD7 chimeric antigen receptor (CAR) T-cell safety and efficacy in patients with r/r T-ALL.METHODSIn this single-center, phase I trial, we administered anti-CD7 CAR T cells, manufactured from either previous stem-cell transplantation donors or new donors, to patients with r/r T-ALL, in single infusions at doses of 5 × 105or 1 × 106(±30%) cells per kilogram of body weight. The primary end point was safety with efficacy secondary.RESULTSTwenty participants received infusions. Adverse events including cytokine release syndrome grade 1-2 occurred in 90% (n = 18) and grade 3-4 in 10% (n = 2), cytopenia grade 3-4 in 100% (n = 20), neurotoxicity grade 1-2 in 15% (n = 3), graft-versus-host disease grade 1-2 in 60% (n = 12), and viral activation grade 1-2 in 20% (n = 4). All adverse events were reversible, except in one patient who died through pulmonary hemorrhage related to fungal pneumonia, which occurred at 5.5 months, postinfusion. Ninety percent (n = 18) achieved complete remission with seven patients proceeding to stem-cell transplantation. At a median follow-up of 6.3 months (range, 4.0-9.2), 15 remained in remission. CAR T cells were still detectable in five of five patients assessed in month 6, postinfusion. Although patients' CD7-positive normal T cells were depleted, CD7-negative T cells expanded and likely alleviated treatment-related T-cell immunodeficiency.CONCLUSIONAmong 20 patients with r/r T-ALL enrolled in this trial, donor-derived CD7 CAR T cells exhibited efficient expansion and achieved a high complete remission rate with manageable safety profile. A multicenter, phase II trial of donor-derived CD7 CAR T cells is in progress ( NCT04689659 ).

Pan and colleagues report one of the first prospective evaluations of planned sequential chimeric antigen receptor (CAR) T-cell therapy targeting CD19 and then CD22 in a phase 1 trial, indicating acceptable toxicity and encouraging durable efficacy in pediatric patients with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL).

Rationale: Viruses are the most common pathogens that can cause infection-related non-recurrent death after transplantation, occurring mostly from the early stages of hematopoietic stem cell transplantation (HSCT) to within 1 year after transplantation. Human coronavirus (HCoV)-NL63 is a coronavirus that could cause mortality among patients with underlying disease complications. Serological tests are of limited diagnostic value in immunocompromised hosts and cases of latent infection reactivation. In contrast, macro-genomic high-throughput (DNA and RNA) sequencing allows for rapid and accurate diagnosis of infecting pathogens for targeted treatment. Patient concerns: In this report, we describe a patient who exhibited acute B-lymphocytic leukemia and developed complicated pulmonary HCoV-NL63 infection after a second allogeneic HSCT (allo-HSCT). Six months after the second allo-HSCT, he developed sudden-onset hyperthermia and cough with decreased oxygen saturation. Chest computed tomography (CT) suggested bilateral multiple rounded ground-glass opacities with the pulmonary lobules as units. Diagnoses: HCoV-NL63 was detected by metagenomic next-generation sequencing (NGS), and HCoV-NL63 viral pneumonia was diagnosed. Interventions: The treatment was mainly based on the use of antiviral therapy, hormone administration, and gamma-globulin. Outcomes: After the therapy, the body temperature returned to normal, the chest CT findings had improved on review, and the viral copy number eventually became negative. Lessons: The latest NGS is an effective method for early infection diagnosis. The HCoV-NL63 virus can cause inflammatory factor storm and alter the neutrophil-to-lymphocyte ratio (NLR). This case suggests that the patient's NLR and cytokine levels could be monitored during the clinical treatment to assess the disease and its treatment outcome in a timely manner.

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2–99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

INTRODUCTION The phase 1/2 clinical trials of CD19 CAR T-cell that led to US Food and Drug Administration approval excluded patients with central nervous system (CNS) involvement, due to concerns for CAR T-cell related neurotoxicity. The clinical trial (ChiCTR1800014457) examined efficacy and safety of sequential different B cell antigen-targeted CAR T-cell therapy for pediatric refractory/relapsed (r/r) Burkitt Lymphoma with secondary CNS involvement. METHODS Twenty-three patients up to 18 years of age with r/r Burkitt Lymphoma were enrolled between January 17, 2018 and November 8, 2019 including 10 patients with secondary CNS involvement. All enrolled patients received the first mCD19 CAR T-cell infusion. The patients who achieved an ongoing complete response (CR) did not receive further therapy; for patients who retained a partial response (PR) or no response (NR) until mCD19 CAR T cells became undetectable in PB by flow cytometry (FCM), the second hCD22 CAR T-cell infusion was initiated so that it did not prematurely interfere with the antitumor effect of mCD19 CAR T cells, and could prevent disease progression in the setting of continuous mCD19 CAR T-cell contraction; and for patients who had progressive disease (PD) or relapsed disease (RD) after responding to mCD19 CAR T cells, considering that the tumor had already produced immune escape from mCD19 CAR T cells, regardless of whether there were mCD19 CAR T cells detectable in PB by FCM, the second hCD22 CAR T-cell infusion was immediately initiated. After the second hCD22 CAR T-cell infusion, the patients who retained a PR until hCD22 CAR T cells became undetectable in PB by FCM received the third hCD20 CAR T-cell infusion; for patients who suffered relapses after achieving the first CR (CR1) following the first infusion and then attained the second CR (CR2) by the second infusion, the third hCD20 CAR T-cell infusion was given as consolidation therapy after loss of hCD22 CAR T cells detectable in PB by FCM, and the patients who had PD after having obtained a PR immediately received the third hCD20 CAR T-cell infusion even if hCD22 T cells were still detectable in PB by FCM. After the third hCD20 CAR T-cell infusion, the patients who retained a PR until hCD20 CAR T cells became undetectable in PB by FCM were given the fourth hCD19 CAR T-cell infusion. RESULTS The median time from the last infusion to cutoff date was 17 months (range, 15 to 23). At 18 months after the last infusion, all patients had an estimated PFS rate of 78% (95% CI, 55 to 90) and OS rate of 83% (95% CI, 60 to 93). Through mCD19 CAR T-cell expansion in the cerebrospinal fluids (CSF) of ten patients with CNS involvement, 8 patients achieved a CR, and 2 PR. Among the 8 patients who achieved a CR, 4 patients (P5, P15, P16, and P19) maintained an ongoing CR, whereas 4 patients (P7, P11, P17, and P21 ) developed a RD at m6, m2, m3, and m1.5 after infusion. P17 died of rapid intracerebral mass (ICM) progression, which resulted in elevated intracranial pressure (ICP), a contraindication to further CAR T-cell therapy. Two patients (P7 and P21) with CNS RD, P11 with bone marrow RD, and 2 patients (P13 and P20) with PR received the second hCD22 CAR T cells infusion. Among 5 patients experiencing 2 cycles of CAR T-cell infusion, 4 patients (P7, P13, P20, and P21) had hCD22 CAR T-cell expansion in CSF. P13 achieved an ongoing CR1; P20 and P21 died of ICM progression, who did not proceed with the third hCD20 CAR T-cell infusion due to elevated ICP and loss of CD20 in tumor cells, respectively; P7 and P11 achieved a CR2, who underwent the third hCD20 CAR T cells infusion, and then had hCD20 CAR T cells expansion in CSF. P11 achieved an ongoing CR2; P7 had a CNS RD again at m3 after obtaining a CR2 and finally died of rapid disease progression. Six patients achieved ongoing CR after the last infusion. The estimated 18-month PFS rate of 60% (95% CI, 25 to 83) in the patients with CNS involvement was not significantly lower than 92% (95% CI, 56 to 99) in the patients without CNS involvement (p=0.28 ). Grade 3 neurotoxicity occurred in 30% (3/10) of patients with CNS involvement. All adverse events were reversible. Statistically significant difference was not observed in the severity of neurotoxicity between patients with and without CNS involvement (p= 0.62). CONCLUSION Sequential CAR T-cell therapy may result in a durable response and is safe in pediatric r/r Burkitt lymphoma. Patients with secondary CNS involvement may benefit from sequential CAR T-cell therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Chimeric antigen receptor T-cell (CART) Therapy has induced high complete remission (CR) in the patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but the median event-free survival is only 6.1 months (Park JH. et al. NEJM 2018). To achieve durable leukemia-free survival (LFS), it may helpful to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CR with CART therapy. Objective: In current study, safety and long-term outcomes of allo-HSCT after CART therapy in r/r B-ALL were assessed and risk factors for overall survival (OS) and LFS were analyzed. Methods: Between June 2017 to June 2020, 137 patients with r/r B-ALL who achieved CR with CD19 or CD22 CART then bridge to allo-HSCT in our hospital were evaluated. The median age was 9.7 years (1.8-56). Bone marrow (BM) samples were collected to detect somatic gene mutations of leukemic cells. Blood samples from patients, parents, and potential related donors were collected to analyze germline mutations of hematological and immunological hereditary predisposition genes. The disease status before transplant was minimal residual disease (MRD) negative in 114 (83.2%) patients and MRD positive in 23 (16.8%) patients. The median time from CART infusion to allo-HSCT was 51 days (29-94). The types of transplant were haploidentical (105, 76.6%), unrelated (26, 19.0%), and identical siblings (6, 4.4%). Myeloablative reduced-toxicity conditioning (RTC) with either total body irradiation (TBI)/fludarabine-based (84, 61.3%) or busulfan (BU) /fludarabine-based (53, 38.7%) were applied. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. Results: Full donor engraftment was achieved in all patients. The median time for neutrophil and platelet engraftment was 15 days (9-21) and 13 days (5-33). With a median follow-up of 653 days (481-783), two-year OS was 74.1% (64.4-81.5) and two-year LFS was 64.4% (54.6-72.6). The incidences of grade Ⅱ to Ⅳ and severe acute GVHD (aGVHD) were 27%, 14.5%, respectively. Twenty-five (18.2%) cases developed chronic GVHD (cGVHD) (limited 18, extensive 7). The incidences of CMV and EBV viremia were 20.1%, 12.5%. Hemorrhagic cystitis was found in 32 (22.2%) cases. Thirty-six (26.3%) cases relapsed after transplant, 28/114 (24.6%) in MRD negative cohort and 8/23 (43.5%) in MRD positive cohort. Thirty (21.9%) patients died (relapsed 22, infection 5, multiple organ failure 2, thrombotic microangiopathy 1). Transplant-related mortality was only 5.8%. Total 137 patients with 99 kinds of somatic mutations, the most frequent 15 mutations were TP53, CYP2C19, KRAS, NRAS, PTPN11, CREBBP, KMT2D, KIT, NR3C1, STK11, FLT3, NF1, NUDT15, ABL1, ADAMTS13. LFS was significantly reduced in patients with TP53, KIT, NF1 mutation (P < 0.0001; P < 0.025; P < 0.003). Total 114 evaluable patients with 93 kinds of hematological and immunological hereditary predisposition gene mutations, the most frequent 15 mutations were BLTA, SERPINE1, CYP2C19, F7, TP53, DPYD, MLH1, TNFAIP3, YARS2, HIFIA, ATM, EP300, KIT, ADAMTS13, BRCA2. LFS was significantly reduced in patients with germline EP300 mutation (P < 0.00027). Univariate analysis showed that BU-based regimen (P=0.0194), somatic TP53 mutation (P= 0.002), fungal infection (P= 0.0217), viral infection (P=0.0187) and MRD positive before transplant (P= 0.0227) were adverse factors of OS. The negative factors of LFS were BU-based regimen (P=0.0139), germline EP300 mutation (P Conclusions: Our large series and long-term outcomes have demonstrated that LFS and OS have been improved remarkably in r/r B-ALL who achieved CR with CART then quick bridge to RTC allo-HSCT. Our study has shown the somatic and germline mutation profiles in r/r/ B-ALL and found that somatic TP53 mutation and germline EP300 mutation are negative factors for LFS. TBI-based regimen , MRD negative before transplant and aGVHD have positive impact on LFS, and fungal infection, somatic TP53 and germline EP300 have negative impact on LFS. Disclosures No relevant conflicts of interest to declare.

Background: Epstein-Barr virus (EBV) positive hematological malignancies and EBV-associated hemophagocytic lymphohistiocytosis (HLH) are serious illness that is related with chronic active EBV infection (CAEBV). Recently, more and more immune-related gene mutations have been identified to associate with the EBV positive hematological diseases. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for above diseases. Aim: In present clinical study, the germline gene mutations associated with EBV positive hematological malignancies and EBV positive HLH were analyzed and the outcomes of allo-HSCT in those diseases were investigated. Methods: Between January 2018 and June 2021, 7 patients with EBV positive hematological diseases (lymphoma 3, aggressive NK-cell leukemia (ANKL) 1, secondary HLH 3) who received allo-HSCT in our hospital were included. Blood samples from patients, parents and potential related donors were collected before transplantation and whole exome sequencing of the blood DNA samples was performed to detect and analyze their relevance with the clinical features before and after allo-HSCT. The median age was 23 (5-31) years old. The median disease course before transplant was 6 (3-38) months. Before allo-HSCT, 3 patients (HLH 1, lymphoma 1, ANKL 1) were in non-remission (NR), 2 patients with lymphoma were in partial remission (PR) and the other two patients with HLH were in complete remission (CR). Five patients received allo-HSCT from haploidentical donors and 2 patients from unrelated donors (HLA 10/10, 9/10 matched). Myeloablative conditioning regimen with busulfan (0.8mg/kg iv q6h, 3 days)/etoposide (200mg/m 2, 3 days)/fludarabine (30mg/m 2, 5 days) /rabbit anti-T-cell globulin was used. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. Results: All patients achieved durable engraftment. Plasma EBV-DNA was positive in three patients (1200, 880, 84000 copies/ml, respectively) before transplant and has been negative in all patients after transplant. One patient had gastrointestinal bleeding caused by EBV enteritis. Two patients had CMV viremia. Three patients developed grade III acute GVHD and two had extensive chronic GVHD. All GVHD was resolved with immune-suppressants. With the median follow-up time of 21 (7-36) months, all patients are alive and free of their diseases. Seven disease-related gene mutations were detected including FANCA p.S1088F, TGFBR2 p.T315M, IRF7 p.K446R, PRF1 p.R4C, MPEG1 p.P316S, STX11 p.R49Q, and UNC13D p.G863D. The first 5 gene mutations were detected in the patients with lymphoma and ANKL, and the last 2 gene mutations were detected in the patients with HLH. FANCA is associated with bone marrow failure and the other genes are associated with primary immunodeficiency, all of these gene mutations have been reported and affect in part the function of the genes. In particular, STX11 p.R49Q and UNC13D p.G863D have also been reported in HLH patients and the NK cell activity of these patients is lower than normal levels. Apart from the defined gene mutations in primary HLH, recently, many new immunodeficiency gene mutations have been reported to associate with EBV positive malignancies and HLH which suggests immunodeficiency might play a role in the etiology of the diseases. The gene mutations we reported here might be the main gene mutations associated with disease susceptibility and infection risk. Conclusion: Our preliminary results have shown the gene mutation profile related to EBV positive hematological malignancies and HLH. With our transplant protocol, durable reconstitution of hematopoiesis and immune from healthy donors has been achieved successfully which has resulted in 100% disease-free survival and no transplant-related death. Allo-HSCT is a safe and effective curative modality for EBV positive hematological malignancies and HLH. Disclosures No relevant conflicts of interest to declare.

Acute graft-versus-host disease (aGVHD) is one of major complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Genetic polymorphisms of interleukin-7 receptor (IL-7R) have an impact on transplant-related mortality (Z Shamim et al. BMT 2006). Our previous studies indicated that hematological and immunological hereditary predisposition genes have potential influence on major complications of allo-HSCT in patients with hematological malignancies (unpublished data). In present study, the influence of hematological and immunological hereditary predisposition genes on aGVHD after allo-HSCT in the patients with hematological malignancies was investigated. Between February 2018 and August 2019, 92 patients with hematological malignancies who underwent allo-HSCT in Beijing Boren Hospital were enrolled. The median age was 17 (1 to 45) years old. The diagnoses included acute myeloid leukemia (37, 40.2%), acute lymphoblastic leukemia (36, 39.1%), non-Hodgkin's lymphoma (13, 14.1%), and myelodysplastic syndrome (6, 6.5%). The disease status before transplant was complete remission in 54 (58.7%), non-remission in 23 (25%), and relapse in 15 (16.3%). Before transplants, blood samples were collected from the patients, their parents and potential related donors to detect hematological and immunological hereditary predisposition genes with whole exon sequencing and validation by sanger sequencing. The donors were from identical siblings (4, 4.3%) or matched unrelated volunteers (8, 8.7%) or haploidentical family members (80, 86.9%). Myeloablative conditioning regimens with either total body irradiation/fludarabine-based or busulfan/fludarabine-based were applied. Anti-thymocyte globulin was used in haploidentical and unrelated transplants. GVHD prophylaxis was with cyclosporine, short-term methotrexate and mycophenolate mofetil. Methylprednisolong 1-2mg/kg daily was used for aGVHD treatment and basiliximab was administrated in severe aGVHD. Forty-eight patients (52.2%) developed aGVHD as aGVHD group and 44 patients (47.8%) have no aGVHD as non-aGVHD group. IL7R gene heterozygous mutations in recipients significantly increased overall aGVHD and also severe aGVHD after allo-HSCT. IL7R gene heterozygous mutations were found in 7/48 patients in aGVHD group, but 0/44 in non-GVHD group (P = 0.041). The incidence of grade III-IV aGVHD in patients with IL7R gene heterozygous mutations was significantly higher than that without IL7R gene heterozygous mutations. (5/7 vs. 7/41, P=0.007). Among the patients with IL7R gene heterozygous mutations in aGVHD group, 5/7 patients (71.4%) had grade III-IV aGVHD. Three of them with grade III aGVHD had the same heterozygous mutation site: c.c1241t; p.t414m and rest 2 patients with grade IV aGVHD had the same heterozygous mutation site: c.g314a; p. S105n. Our preliminary results have shown that IL7R gene heterozygous mutations in recipients have significantly increased the risk of overall aGVHD and severe aGVHD after allo-HSCT in the patients with hematological malignancies. Larger sample size is needed to address this issue. Disclosures No relevant conflicts of interest to declare.

Chromosome abnormalities and gene variants are important factors in prognosis of the patients with hematological malignancies. Our previous study showed that some germline gene mutations-related to hematological and immunological disorders may have negative impact on the complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In current study, the influence of both hematological and immunological hereditary predisposition gene variants and tumor gene variants on the outcomes of allo-HSCT in patients with hematological malignancies was studied. Between January 2018 and June 2020, 164 patients with hematological malignancies who underwent allo-HSCT in our hospital were analyzed. The median age was 14 (1 to 67) years old. The diagnosis included acute myeloid leukemia (n=68, 41.5%), acute lymphoblastic leukemia (n=67, 40.8%) and non-Hodgkin's lymphoma (n=29, 17.7%). The disease status before transplant was CR1 in 49 cases (29.9%), CR2 in 63 cases (38.4%), PR in 22 cases (13.4%), and NR in 30 cases (18.3%). Donors were from haploidentical family members (n=125, 76.2%) or identical siblings (n=18, 11.0%) or unrelated volunteers (n=21, 12.8%). Myeloablative conditioning regimens with either total body irradiation/fludarabine-based or busulfan/fludarabine-based were applied. Anti-thymocyte globulin was used in haploidentical and unrelated transplants. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine, short-term methotrexate and mycophenolate mofetil. Before transplant, blood samples from patients, their parents and potential related donors were collected to analyze for more than 700 kinds of hematological and immunological hereditary predisposition genes with whole exon sequencing and validation by sanger sequencing. The average sequencing depth was 150×. At diagnosis or relapse, bone marrow samples from patients were obtained to detect for 339 kinds of tumor genes by Illumina sequencing. The average sequencing depth was more than 3000×. With the median follow-up 12.6 (11.2 to 17.0) months, 105 patients (64.0%) achieved durable remission after allo-HSCT. Forty-seven patients (28.7%) relapsed. Twelve patients (7.3%) died. Total 191 immunodeficiency-related hereditary predisposition gene variants were identified in this cohort (average 3.5 gene variants per patient; range, 0-10). Twenty-six of them were recurrent more than 6 times that including TYK2, IFIH1, CFTR, LRBA, IL7R, POLE, RNF31, NLRP12, TTC7A, ATM, CARD14, CHD7, NOD2, TNFRSF13B, BLB,CFB, EPG5, C8A, C8B, CFH, IRF, MSH6, NCF2, NFAT5, PMS2 and ST1M1.IFIH1and IL10RB gene variants were poor factors for relapse post-transplant (P=0.006, P=0.007). The functions of these genes involve in combined immunodeficiency, autoinflammatory disease, complement deficiency, immune deficiency, T-cell dysfunction and antibody deficiency. Total 153 tumor gene variants were identified in this patient series (average 1.93 gene variants per patient; range, 0-16). Seventeen of them were recurrent more than 4 times that including TP53, TPN11, KIT, FLT3, NRAS, NUDT15, STK11, CREBBP, NPM1, DNAH9, DNMT3A, IDH2, HEK2, KRAS, KMT2C, NOTCH1 and NR3C1. TP53, KRAS and NUDT15 gene variants were poor factors for relapse after allo-HSCT (P=0.000025, P=0.0082, P=0.000018). Hemophagocytic lymphohistiocytosis (HLH)-related genes variants were found in 9 patients (5.5%). Frequent HLH-related gene variants were in CD27, PRF1, STX11, and UNC13D. Fanconi anemia (FA)-related gene variants were seen in 11 patients (6.7%). Common FA-related gene variants were in BRCA1, BRCA2, BRIP1, FANCA, FANCC, FANCF, FANCM and FANCG. Significantly higher incidences of acute GVHD (aGVHD) and/or infections were noted in the patients with HLH and/or FA-related gene variants compared with those without HLH and FA-related gene variants (p=0.019). Our results have shown that both inherited and acquired hematological and immunological-related gene variant profiles in the patients with hematological malignancies and some recurrent gene variants (IFIH1 and IL10RB; TP53, KRAS and NUDT15) have negative impact on the outcomes of allo-HSCT including leukemia-free survival, aGVHD and infections. Keywords: allogeneic hematopoietic stem cell transplant, gene expression, relapse, hematological malignancy Disclosures No relevant conflicts of interest to declare.

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2-99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

The clinical utility of Traditional Chinese Medicine (TCM) herbs/roots extracts in osteoporosis (OP) and osteoarthritis (OA) has been described in multiple reports, but there have been few studies of TCM for preventing bone loss and cartilage degradation simultaneously. Six-month-old female Sprague-Dawley rats each were subjected to ovariectomized (OVX) or sham surgery and treated orally once daily with herbal extracts or vehicle. Body weight was recorded weekly, and blood samples were collected from fasting animals at different time points. Biochemical markers of bone resorption and cartilage degradation were analyzed. Changes in bone mineral density and calcium content were determined in the femoral center and femoral telocentric end of rats. Out of 56 TCM herbs/roots extracts, only kudzu root demonstrated consistent joint protective effects. OVX resulted in a marked increase in bone resorption and cartilage degradation, which could be significantly reversed by kudzu after three weeks of treatment. Compared to vehicle, kudzu induced a significant increase in bone mineral density in the femoral center and femoral telocentric end, and calcium content. The results show that kudzu exerts direct effects on articular cartilage in the OVX rat and can effectively prevent the acceleration of cartilage degradation induced by ovariectomy. Moreover, kudzu has demonstrated positive effects on metabolic health (cause a weight reduction) and may represent a possible treatment for OP and OA with high body mass index. Further studies are needed to investigate the potential effects of kudzu root in postmenopausal women.

The clinical utility of Traditional Chinese Medicine (TCM) herbs/roots extracts in osteoporosis (OP) and osteoarthritis (OA) has been described in multiple reports, but there have been few studies of TCM for preventing bone loss and cartilage degradation simultaneously. Six-month-old female Sprague-Dawley rats each were subjected to ovariectomized (OVX) or sham surgery and treated orally once daily with herbal extracts or vehicle. Body weight was recorded weekly, and blood samples were collected from fasting animals at different time points. Biochemical markers of bone resorption and cartilage degradation were analyzed. Changes in bone mineral density and calcium content were determined in the femoral center and femoral telocentric end of rats. Out of 56 TCM herbs/roots extracts, only kudzu root demonstrated consistent joint protective effects. OVX resulted in a marked increase in bone resorption and cartilage degradation, which could be significantly reversed by kudzu after three weeks of treatment. Compared to vehicle, kudzu induced a significant increase in bone mineral density in the femoral center and femoral telocentric end, and calcium content. The results show that kudzu exerts direct effects on articular cartilage in the OVX rat and can effectively prevent the acceleration of cartilage degradation induced by ovariectomy. Moreover, kudzu has demonstrated positive effects on metabolic health (cause a weight reduction) and may represent a possible treatment for OP and OA with high body mass index. Further studies are needed to investigate the potential effects of kudzu root in postmenopausal women.

Introduction: The development of hematological malignancies may be relevant to hereditary predisposition and harmful external environment. Germ line predisposition to myeloid neoplasms has been incorporated in the WHO 2016 classification of myeloid neoplasms. Hereditary predisposition genes may also play a certain role in the complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Objective: In present clinical study, we investigate whether hematological and immunological hereditary predisposition genes have influence on the outcomes of allo-HSCT in patients with hematological malignancies. Methods: Between January 2018 and May 2019, ninety-one patients with hematological malignancies who underwent allo-HSCT with the same protocols in our hospital were enrolled. The median age was 20 (2 to 66) years old. Male to female was 1.53:1. The diagnosis included AML (n=32, 35.2%), ALL (n=47, 51.6%) and NHL (n=12, 13.2%). The disease status before transplants was CR1 in 31 cases (34.1%), CR2 in 45 cases (49.5%), PR in 5 cases (5.5%), and NR in 10 cases (11.0%). Hematological and immunological hereditary predisposition genes of the patients, their parents and potential related donors were prospectively detected before transplant with whole exon sequencing and validation by sanger sequencing. Donors were from haploidentical family members (n=74, 81.3%) or identical siblings (n=6, 6.6%) or unrelated volunteers (n=11, 12.1%). Myeloablative conditioning regimens with either TBI/Fludarabine or Busulfan/Fludarabine were applied. Anti-thymocyte globulin (ATG) was used in haploidentical and unrelated transplants. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine, short-term methotrexate and mycophenolate mofetil. Prevention of fungal, pneumocystis carinii and herpes virus infections was routinely administrated. Chi-square test was used to analyze whether hereditary predisposition genes were associated with major complications after allo-HSCT. The study used the IBM SPSS Statistics software for analysis. P value Results: Thirty-one (34.1%) cases carried primary hemophagocytic lymphohistiocysis (HLH) related gene variants and the average number of gene variants was 1.2 (range 1 to 2). More frequent HLH gene variants were in UNC13D, LYST, PRF1, AP3B1 and STX11. Forty-four (48.4%) cases carried Fanconi anemia (FA) related gene variants and the average number of gene variants was 1.27 (range 1 to 3). More frequent FA gene variants were in BRCA2, FANCA, PALB2, FANCD2, BRCA1, FANCL, FANCI and SLX4. Ninety (98.9%) cases carried immunodeficiency related gene variants and the average number of gene variants was 3.25 (range 1 to 10). More frequent immunodeficiency gene variants were in IFIH1, CHD7, TYK2, LRBA, NFAT5, IL7R, NLRP12, POLE, TNFRSF13B, ATM, IGLL1, ORAI1, RNF31, C8A, CBLB, IRF8, LIG1, NCF2, NCF4, STIM1 and TCF3 .The incidence of acute GVHD (aGVHD) in patients carried HLH gene variants was significantly higher than that without HLH gene variants (45.1% vs. 21.6%, p=0.020). Bacterial infections occurred more frequent in patients with FA gene variants than that without FA gene variants (47.7% vs. 21.3%, p = 0.008). There was a trend of higher relapse rate in patients with more than 3 immunodeficiency gene variants than that with less than 3 immunodeficiency gene variants but without statistical significance (23.8% vs. 11.4%, p > 0.05). Conclusions: Our preliminary results have shown that hematological and immunological hereditary predisposition genes have potential influence on major complications of allo-HSCT in patients with hematological malignancies. HLH gene variants increase the risk of aGVHD, and FA gene variants increase bacterial infections after allo-HSCT. The patients with multiple immunodeficiency related gene variants may increase relapse rate post-transplant. A larger cohort and longer follow-up are needed to address this issue. Disclosures No relevant conflicts of interest to declare.

Objective Cathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or ankylosing spondylitis. Methods Presently there are no robust assays for the assessment of active cathepsin K in serum, and therefore an ELISA specifically detecting the N-terminal of the active form of cathepsin K was developed. Results The assay was technically robust, with a lowest limit of detection (LOD) of 0.085 ng/mL. The average intra- and inter-assay CV% were 6.60% and 8.56% respectively. The dilution recovery and spike recovery tests in human serum were within 100 ± 20% within the range of the assay. A comparison of latent and active cathepsin K confirmed specificity towards the active form. Quantification of the levels of active cathepsin K in supernatants of purified human osteoclasts compared to corresponding macrophages showed a 30-fold induction (p In contrast, in serum samples from osteoporotic women on estrogen or bisphosphonate therapy and from ankylosing spondylitis patients no clinically relevant differences were observed. Conclusion In summary, we have developed a robust and sensitive assay specifically detecting the active form of cathepsin K; however, while it monitors osteoclasts with high specificity in vitro, it appears that circulating levels of active cathepsin K do not reflect bone changes under these circumstances.

Background Immobilization-induced loss of muscle mass is a complex phenomenon with several parallels to sarcopenic and cachectic muscle loss. Muscle is a large organ with a protein turnover that is orders of magnitude larger than most other tissues. Thus, we hypothesize that muscle loss and regain is reflected by peptide biomarkers derived from type VI collagen processing released in the circulation. Methods In order to test this hypothesis, we set out to develop an ELISA assay against an type VI collagen N-terminal globular domain epitope (IC6) and measured the levels of IC6 and an MMP-generated degradation fragment of collagen 6, (C6M) in a human immobilization–remobilization study setup with young (n = 11) and old (n = 9) men. They were subjected to 2 weeks of unilateral lower limb immobilization followed by 4 weeks of remobilization including thrice weekly resistance training, using the contralateral leg as internal controls. Subjects were sampled for strength, quadriceps muscle volume and blood at baseline (PRE), post-immobilization (2W), and post-remobilization (4W). Blood were subsequently analyzed for levels of the C6M and IC6 biomarkers. We subsequently tested if there was any correlation between C6M, IC6, or the C6M/IC6 ratio and muscle mass or strength at baseline. We also tested whether there was any relation between these biomarkers and changes in muscle mass or strength with immobilization or remobilization. Results The model produced significant loss of muscle mass and strength in the immobilized leg. This loss was bigger in young subjects than in elderly, but whereas the young recovered almost fully, the elderly had limited regrowth of muscle. We found a significant correlation between IC6 and muscle mass at baseline in young subjects (R2 = 0.6563, p = 0.0045), but none in the elderly. We also found a significant correlation between C6M measured at the 4W time point and the change in muscle mass during remobilization, again only manifesting in the young men(R2 = 0.6523, p = 0.0085). This discrepancy between the young and the elderly may be caused in part by much smaller changes in muscle mass in the elderly and partly by the relative small sample size. Conclusion While we cannot rule out the possibility that these biomarkers in part stem from other tissues, our results strongly indicate that these markers represent novel biomarkers of muscle mass or change in muscle mass in young men. Electronic supplementary material The online version of this article (doi:10.1007/s13539-013-0114-x) contains supplementary material.

Objectives Type V collagen has been demonstrated to control fibril formation. The aim of this study was to develop an ELISA capable of detecting a fragment of type V collagen generated by MMP-2/9 and to evaluate the assay as biomarker for ankylosing spondylitis (AS). Design and methods A fragment unique to type V collagen and generated by both MMP-2/9 cleaved at the amino acid position 1317 (C5M) was selected for ELISA development. 40 AS patients and 40 age-matched controls were evaluated. Results An ELISA detecting C5M with inter- and intra-assay variations of 9.1% and 4.4% was developed. C5M levels were significantly higher in AS patients compared to controls, 229% (p Conclusions This ELISA is the first for detecting type V collagen degradation. AS patients had highly elevated levels of MMP mediated type V collagen degradation. The prognostic and diagnostic values need to be further investigated in additional clinical settings.

Aim: The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100% homologous in the human, rat, and mouse species. Methods: Monoclonal antibodies were raised against selected P4NP 7S specific sequences. Antibodies were screened and a competitive ELISA assay was developed using a selected antibody. The assay was evaluated in relation to technical performance, and in two preclinical liver fibrosis models; the bile duct ligation model (BDL) and the carbon tetrachloride model (CCL4) both performed in rats. Results: A technically robust P4NP 7S ELISA assay using a monoclonal antibody was produced. In the BDL and CCL4 liver fibrosis models it was observed that the P4NP 7S levels were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine.A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice.The lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice.We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.

In joint degenerative diseases, the collagens are degraded by matrix metalloproteinases and protein fragments are released to serum as potential biomarkers.A collagen type II specific neoepitope, CIIM, was identified (…RDGAAG(1053)) by mass spectrometry. Two ELISAs against the neoepitope were developed. CIIM was measured in cartilage explants in the presence or absence of protease inhibitors. CIIM was measured in OA synovial fluid (n=51) and serum (n=156). Knee OA was graded by standard Kellgren-Lawrence (KL) score.The ELISAs showed good technical performance; CV%,13%. CIIM release from cartilage explants was blocked by the MMP inhibitor. CIIM was detected in synovial fluid. Furthermore, serum CIIM levels were significantly higher (P0.05) in those individuals with mild or severe OA than in those with no OA.We developed a new biomarker for joint degenerative diseases, which we demonstrated was derived from MMP-degraded type II collagen.

The alpha1,2-fucosyltransferase family (alpha1,2FT) is the largest family of glycosyltransferases in the genome of the free-living nematode Caenorhabditis elegans, and early evidence suggests that each member may have a unique activity. Here we describe a C. elegans gene (designated CE2FT-2) encoding an alpha1,2FT that has the potential to generate the sequence Fucalpha1-2Galbeta1-3GalNAcalpha-R, which is the H-type 3 blood group structure. The CE2FT-2 cDNA encodes a putative transmembrane protein that shows approximately 42% amino acid identity to a previously cloned C. elegans alpha1,2FT (termed CE2FT-1), but has a very low identity ( approximately 16-20%) to alpha1,2FT sequences in humans, rabbits, and mice. A recombinant form of CE2FT-2 expressed in human 293T cells has a high alpha1,2FT activity toward Galbeta1-3GalNAcalpha-O-pNP, but unexpectedly, the enzyme is inactive toward the acceptor Galbeta-O-phenyl. Thus, CE2FT-2 differs from all other alpha1,2FTs previously described from animals that all utilize Galbeta-O-phenyl. CE2FT-2 is expressed at all stages of worm development, but remarkably, promoter analysis of the CE2FT-2 gene using green fluorescent protein reporter constructs indicates that the CE2FT-2 is expressed exclusively in pharyngeal cells of the worm from embryo to an adult stage. Because pharyngeal cells are known to secrete their glycoconjugates to the nematode surface, these results may indicate that products of CE2FT-2 contribute to interactions of the nematode with its environment or are used as ligands for bacterial attachment. These findings, along with those on other alpha1,2FTs in C. elegans, suggest that each alpha1,2FT in this organism may have a unique acceptor specificity, expression pattern, and biological function.

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.

Background Osteoarthritis (OA) is the most common degenerative joint disease, of which the pathogenesis is inadequately understood. Hypertrophy-like changes have been observed as part of the progression of OA. The aim of the study was to develop and characterize a novel biomarker of chondrocytes hypertrophy and investigate how this marker was associated with cartilage degradation and inflammation in patients with various degrees of OA. Methods A competitive ELISA, C-Col10, applying a well-characterized monoclonal antibody was developed as a biomarker of chondrocyte hypertrophy through measurement of type X collagen (ColX). The levels of C-Col10, C2M (matrix metalloproteinase-derived fragments of type II collagen) and hsCRP (high sensitive C-reactive protein) were quantified by ELISAs in serum of 271 OA patients stratified by Kellgren-Lawrence (KL) score 0–4. Associations between serum levels of the three biomarkers (log transformed) were analyzed by Pearson’s correlation and differences in C-Col10 levels between patients with high and low levels of inflammation measured by hsCRP were analyzed by ANOVA. Results We developed a C-Col10 assay measuring the C-terminus of ColX. We found significantly higher levels of ColX in patients with KL score 2 compared to patients with no radiographic evidence of OA (KL0) (p = 0.04). Levels of ColX were significantly elevated in OA patients with above normal hsCRP levels (p

The alterations of the cytoskeletal actin network have been implicated as a morphological effector in apoptosis. However, studies directly linking actin change to the morphological events in apoptosis are lacking. This study quantitatively examined the effect of actin alteration on the camptothecin (CPT)-induced apoptotic process in HL-60 cells. Actin alteration was induced by two distinctive types of agent: the polymerization-stimulating agent, Jasplakinolide (Jas), and the polymerization-blocking agent, cytochalasin B (CB). The actin polymerization status was measured by two complementary methods: the cell pellet-based DNase I inhibition method, and the individual cell-based quantitative fluorescence image analysis (QFIA) assay. Actin polymerization induced by Jas caused apoptosis directly. By contrast, CB, an actin polymerization-blocking agent, partially inhibited CPT-induced apoptosis. A similar inhibition of the CPT-induced apoptosis response was observed with a more specific actin depolymerization agent, cytochalasin E. The alterations of the actin polymerization status occurred in three sequential steps during the apoptotic process: first polymerization, followed by depolymerization, and finally degradation. However, compared with CPT-induced apoptosis, Jas-induced apoptosis was characterized by pronounced actin polymerization that corresponded morphologically with prominent membrane blebbing, but less apoptotic body formation. Furthermore, DNase I activity, which is normally inhibited by G-actin, was specifically detected in Jas-treated cells. These results show that the regulation of actin polymerization is an important apoptotic morphological effector, whereas the alterations of the actin polymerization status by chemicals have profound effects not only on altering the morphology of apoptotic cells, but on apoptosis induction in HL-60 cells as well.

Background Matrix metalloproteinase-3 (MMP-3) plays an important role in the pathology of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Measurement of active MMP-3 in clinical samples could provide information about progression of rheumatoid diseases, and potentially response to treatment. Hence, we aimed to develop a sensitive assay specifically measuring the active form of MMP-3 (act-MMP-3) both in ex vivo models and in human sera. Methods A monoclonal antibody against the first 6 amino acids of act-MMP-3 was developed, and the specificity was carefully tested by comparing total and active MMP-3. A technically robust act-MMP-3 ELISA was produced. For biological validation, human synovial membrane and human cartilage explant (HEX) culture models were measured and compared by ELISA and immunoblots. For clinical relevance, the serum levels of act-MMP-3 in AS and RA patients before and after anti-TNF-α treatment were evaluated. Results A highly specific and technically robust ELISA detecting act-MMP-3 in serum was developed. The lower limit of detection was 33.7 pg/mL. The dilution and spiking recovery of human serum was within 100 ± 20%. The average intra- and inter-assay variations were 3.1% and 13.5% respectively. High levels of act-MMP-3 expression were observed in human synovial membrane culture and oncostatin M and TNF-α stimulated human cartilage. In a cross-sectional study of both AS and RA patients, serum act-MMP-3 level was correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). In addition, in patients receiving anti-TNF-α treatment, the serum level of act-MMP-3 was significantly reduced compared to baseline level reflecting the anti-inflammatory effects of the treatment. Conclusion We have successfully developed an assay measuring act-MMP-3 in human serum showing correlation to inflammatory markers. Further studies are required to clarify, whether act-MMP-3 can serve as a predictive marker for outcome in chronic rheumatoid disorders.

Aim: The present study describes the assessment of true formation of type III collagen in different pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the N-terminal propeptide of type III collagen (PRO-C3). Methods: The monoclonal antibody was raised against the N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical performance, and as a marker for liver fibrosis and muscle mass using the rat carbon tetrachloride (CCl4) model and a study of immobilization induced muscle loss in humans, respectively. Results: The ELISA was neo-epitope specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model it was observed that serum PRO-C3 were significantly elevated in rats with liver fibrosis as seen by histology (56% elevated in the highest quartile of total hepatic collagen compared to control rats, p

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE(373)) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease.

ObjectiveAlzheimer's disease (AD) is a devastating neurological disease characterized by pathological proteolytic cleavage of tau protein, which appears to initiate death of the neurons. The objective of this study was to investigate whether a proteolytic fragment of the tau protein could serve as blood-based biomarker of cognitive function in AD.MethodsWe developed a highly sensitive ELISA assay specifically detecting an A Disintegrin and Metalloproteinase 10 (ADAM10)-generated fragment of tau (Tau-A). We characterized the assay in detail with to respect specificity and reactivity in healthy human serum. We used samples from the Tg4510 tau transgenic mice, which over-express the tau mutant P301L and exhibit a tauopathy with similarities to that observed in AD. We used serum samples from 21 well-characterized Alzheimer's patients, and we correlated the Tau-A levels to cognitive function.ResultsThe Tau-A ELISA specifically detected the cleavage sequence at the N-terminus of a fragment of tau generated by ADAM10 with no cross-reactivity to intact tau or brain extracts. In brain extracts from Tg4510 mice compared to wt controls we found 10-fold higher levels of Tau-A (pConclusionBased on the hypothesis that tau is cleaved proteolytically and then released into the blood, we here provide evidence for the presence of an ADAM10-generated tau fragment (Tau-A) in serum. In addition, the levels of Tau-A showed an inverse correlation to cognitive function, which could indicate that this marker is a serum marker with pathological relevance for AD.

Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. Versican, an extracellular matrix proteoglycan present in normal and atherosclerotic arteries is a substrate for matrix metalloproteinases (MMPs) present in macrophage rich areas. The aim of the current study was to develop an immunoassay to detect a specific MMP-12 derived versican degradation fragment (VCANM) and assess its potential as a biomarker for extracellular matrix remodelling in atherosclerosis. A mouse monoclonal antibody raised against VCANM was used for the development of a competitive ELISA for detection of the fragment in plasma. VCANM was measured in plasma of patients with different levels of heart diseases. Patients experiencing I) acute coronary syndrome, II) stable ischemic heart disease and III) demonstrating high levels of coronary calcium deposits had significantly higher plasma levels of VCANM compared to a control group of individuals with no detectable coronary calcium deposits. VCANM was also detected by immunohistochemistry in coronary artery sections of patients with different degrees of atherosclerosis. VCANM ability to separate patients with atherosclerotic diseases from healthy individuals suggested VCANM as a potential biomarker for the pathological arterial matrix remodelling associated with atherosclerosis.

Cancer is a heterogeneous disease and consequently an exact diagnosis is as important as the actual therapy. Therefore, identification of novel diagnostic biomarker targets is urgently needed. Physiological and pathological changes are reflected by post-translational modifications of proteins. Each post-translational modification (e.g., proteolytic cleavage) is the result of a specific local process and may produce disease-specific neoepitopes. Neoepitopes have been successfully used as biomarkers in many diseases, and may also serve as promising tools in the development of future diagnostic assays within oncology. By specifically targeting neoepitopes, more information regarding disease-type and -state may be obtained and future research into neoepitopes will provide important and novel means for the diagnosis, prognosis and treatment efficacy in cancer. In this paper, we focus on protein ectodomain shedding and the generation of neoepitopes as future noninvasive (serological) cancer biomarkers. We use the protein ectodomain shedding of the human epidermal growth factor receptor 2, which is associated with breast cancer, as an example. We assess the current status of measuring human epidermal growth factor receptor 2 and discuss how this potentially could be improved. Furthermore, we expand the discussion to include examples of other cancer associated proteins.

Background: The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis. The scope of the present study was to develop a novel enzyme-linked immunosorbent assay (ELISA) for the measurement of a MMP-9 and MMP-12-generated biglycan neo-epitope and to test its biological validity in a rat model of RA and in two rat models of liver fibrosis, chosen as models of ECMR.Results: Biglycan was cleaved in vitro by MMP-9 and -12 and the 344 ′YWEVQPATFR ′353 peptide (BGM) was chosen as a potential neo-epitope. A technically sound competitive ELISA for the measurement of BGM was generated and the assay was validated in a bovine cartilage explant culture (BEX), in a collagen induced model of rheumatoid arthritis (CIA) and in two different rat models of liver fibrosis: the carbon tetrachloride (CCL4)-induced fibrosis model, and the bile duct ligation (BDL) model. Significant elevation in serum BGM was found in CIA rats compared to controls, in rats treated with CCL4 for 16 weeks and 20 weeks compared to the control groups as well as in all groups of rats subject to BDL compared with sham operated groups. Furthermore, there was a significant correlation of serum BGM levels with the extent of liver fibrosis determined by the Sirius red staining of liver sections in the CCL4 model.Conclusion: We demonstrated that the specific tissue remodeling product of MMPs-degraded biglycan, namely the neo-epitope BGM, is correlated with pathological ECMR. This assay represents both a novel marker of ECM turnover and a potential new tool to elucidate biglycan role during the pathological processes associated with ECMR.

Disease heterogeneity is as major issue in Type II Diabetes Mellitus (T2DM), and this patient inter-variability might not be sufficiently reflected by measurements of glycated haemoglobin (HbA1c). Β-cell dysfunction and β-cell death are initiating factors in development of T2DM. In fact, β-cells are known vanish prior to the development of T2DM, and autopsy of overt T2DM patients have shown a 60% reduction in β-cell mass. As the decline in β-cell function and mass have been proven to be pathological traits in T2DM, methods for evaluating β-cell loss is becoming of more interest. However, evaluation of β-cell death or loss is currently invasive and unattainable for the vast majority of diabetes patients. Serological markers, reflecting β-cell loss would be advantageous to detect and monitor progression of T2DM. Biomarkers with such capacities could be neo-epitopes of proteins with high β-cell specificity containing post translational modifications. Such tools may segregate T2DM patients into more appropriate treatment groups, based on their β-cell status, which is currently not possible. Presently individuals presenting with adequately elevated levels of both insulin and glucose are classified as T2DM patients, while an important subdivision of those is pending, namely those patients with sufficient β-cell capacity and those without. This may warrant two very different treatment options and patient care paths. Serological biomarkers reflecting β-cell health status may also assist development of new drugs for T2DM and aid physicians in better characterization of individual patients and tailor individual treatments and patient care protocols.